The cOA synthesis assays were performed as previously described10 (link). In brief, a mixture containing 160 μCi [α-32P]-ATP (PerkinElmer) and 500 μM ATP was incubated with 100 nM LlCsm complex, 200 nM target RNA at 37 °C overnight in a cOA synthesis buffer containing 33 mM Tris-acetate pH 7.6 (at 32 °C), 66 mM potassium acetate, 10 mM MgCl2. The reaction products were heat-denatured at 95 °C for 10 min and centrifuged at high speed. The supernatant was mixed with formamide dye, resolved by 8 M Urea, 24% PAGE gels in 1× TBE running buffer at 80 V for 240 min. The gels were carefully sandwiched between non-porous cellophane sheet and a porous gel drying sheet and dried for 120 min, developed for 30 min using Phosphor Screen (GE Healthcare Life Science), and visualized using the Typhoon Gel Imaging System (GE Healthcare Life Science).
To determine which cOAs are produced by LlCsm by mass spectrometry, the same reaction was carried out without [α-32P]-ATP. The reaction products were heat-denatured at 95 °C/10 min and centrifuged at high speed. The supernatant was analyzed by mass spectrometry on an Agilent 6230 TOF-MS with the Agilent Mass Hunter Workstation Software TOF 6500 series in positive ion mode. Spectrum was analyzed using Agilent Mass Hunter Qualitative Analysis Navigator v.B.08 and visualized using GraphPad Prism.
To determine which cOAs are produced by LlCsm by mass spectrometry, the same reaction was carried out without [α-32P]-ATP. The reaction products were heat-denatured at 95 °C/10 min and centrifuged at high speed. The supernatant was analyzed by mass spectrometry on an Agilent 6230 TOF-MS with the Agilent Mass Hunter Workstation Software TOF 6500 series in positive ion mode. Spectrum was analyzed using Agilent Mass Hunter Qualitative Analysis Navigator v.B.08 and visualized using GraphPad Prism.